Defining Suitable Reference Genes for qRT-PCR in Plagiodera versicolora (Coleoptera: Chrysomelidae) under Different Biotic or Abiotic Conditions

Abstract

Plagiodera versicolora (Coleoptera: Chrysomelidae) is one of the most destructive pests of the Salicaceae worldwide, which has established complex interactions with surrounding organisms. Uncovering the molecular mechanisms of some antagonistic interactions would facilitate the development of environmentally friendly pest insect management strategies. Suitable reference genes are essential for reliable qPCR and gene expression analysis in molecular studies; however, a comprehensive assessment of reference genes in P. versicolora is still lacking. In this study, the stability of seven housekeeping genes (including Actin, EF1A, α-tubulin, RPL13a, RPS18, RPL8 and UBC) was investigated under both biotic (developmental stages, tissues, sex and pathogen treatment) and abiotic (RNA interference treatment, temperature treatment) conditions. The geNorm, NormFinder, BestKeeper, and ΔCt programs were used to analyze gene expression data. The RefFinder synthesis analysis was applied to suggest a handful of appropriate reference genes for each experimental condition. RPS18 and EF1A were the most reliable reference genes in different development stages; RPS18 and RPL8 were most stable in female and male adults, different tissues, different temperatures, and pathogen treatment; α-tubulin and RPL13a were most stable after dietary RNAi treatment. The research provides a strong basis for future research into the molecular biology of P. versicolora.